Affinity chromatography of staphylococcal nuclease and bovine pancreatic ribonuclease as well as several ribonuclease derivatives on columns of specific nucleotide inhibitors bound to Sepharose 4B has permitted determination of nucleotide binding constants. Elutions are performed with varying concentrations of active site ligands and MLAB computer data fits are made to derived equations describing the competing equilibria between matrix bound nucleotide and free ligand for enzyme binding.